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Chemistry of Food Additivesand Preservatives

Chemistry of Food A

dditives and Preservatives

Chemistry of Food Additives and Preservatives

Food additives are chemicals or ingredients that are added to food during processing to improve quality, flavour, appearance or nutritional value, or to prevent chemical or microbial spoilage. The most common types of additives are preservatives, colourants, sweeteners, flavourings, emulsifiers, thickeners and stabilisers. Adding new ingredients to a food has an effect upon its chemistry and structure as well as its sensory characteristics. Additives are usually characterised by where they come from (for example, whether they are natural or synthetic), by their purpose (such as improving shelf life) and the risks associated with them (such as their toxicity, and any side effects upon the consumer). Although in recent years the trend in consumer marketing has been to trumpet a lack of additives and preservatives, with ‘artificial ingredients’ commonly seen in a negative light, there nevertheless remains a wide variety of additives and preservatives that are crucial both to producers and consumers, without which the quality of the food would suffer.

Chemistry of Food Additives and Preservatives is an up-to-date reference guide to the wide range of different types of additives used in the food industry today. It looks at the processes involved in adding preservatives and additives to foods, and the mechanisms and methods used. The book provides full details about the chemistry of each major class of food additive, showing the reader not just what kind of additives are used and what their functions are, but also how they work, and how they may have multiple functionalities. This book also covers numerous new additives currently being introduced, how the quality of these is ascertained, and how consumer safety is ensured.

Chemistry of Food Additives and Preservatives is an ideal reference for food chemists, food safety specialists and agencies, food processors who are working with additives and preservatives, and food regulators and policy makers. Written in an accessible style and covering a broad range of food additives and preservatives, the book offers an in-depth analysis of the chemical interactions of food additives and preservatives with the natural composition of the foods to which they are added. It is a unique and ground-breaking treatment of a topic vital to both the food industry and the researcher.

About the Author

Dr Titus A. M. Msagati is a Senior Lecturer in the Department of Applied Chemistry at the University of Johannesburg, South Africa.

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Titus A. M. MsagatiMsa

gati

Titus A. M. Msagati

9 781118 274149

ISBN 978-1-118-27414-9

Msagati_Chemistry_9781118274149_hb.indd 1 31/08/2012 14:03


Titus A. M. Msagati, B.Sc. (Hons), MSc, Ph.D., CChem, MRSCDepartment of Applied ChemistryUniversity of JohannesburgRepublic of South Africa

A John Wiley & Sons, Ltd., Publication

This edition first published 2013 C© 2013 by John Wiley & Sons, Ltd.

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Library of Congress Cataloging-in-Publication Data

Chemistry of food additives and preservatives / Titus A. M. Msagati.p. cm.

Includes bibliographical references and index.ISBN 978-1-118-27414-9 (hardcover : alk. paper) 1. Food additives. 2. Food preservatives.

3. Food–Analysis. 4. Food–Composition. I. Msagati, Titus A. M.TX553.A3C455 2012641.3′08–dc23

2012009754

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Contents

Preface ixIntroduction xList of Abbreviations xiii

1 Antioxidants and Radical Scavengers 11.1 Chemistry of free radicals and antioxidants 11.2 Types of antioxidants 41.3 Efficacy of different antioxidants 71.4 Action mechanisms of antioxidants 91.5 Structure–activity relationship of antioxidants 111.6 Factors affecting antioxidant activity 141.7 Quality assessment of dietary antioxidants 151.8 How safe are food antioxidants? 231.9 Summary 25

References 25Further reading 31

2 Emulsifiers 332.1 Mechanisms of food emulsifiers 332.2 The role of emulsifiers in foods 352.3 Classification of emulsifiers 372.4 Types of food emulsifiers 382.5 Quality and analysis of food emulsifiers 582.6 Foods containing emulsifiers 60


3 Stabilisers, Gums, Thickeners and Gelling Agents as Food Additives 673.1 Introduction to stabilisers, thickeners and gelling agents 673.2 Polysaccharides 683.3 Protein-based food stabilisers 773.4 Quality control of food stabilisers and thickeners 783.5 Analytical methods 78


4 Sweeteners 834.1 Introduction to sweeteners 834.2 Properties of sweeteners 84

vi Contents

4.3 Intense sweeteners in foods 864.4 Bulk food sweeteners 924.5 Quality assurance and quality control 954.6 Analytical methods 98


5 Fragrances, Flavouring Agents and Enhancers 1025.1 Introduction to flavours and flavouring agents 1025.2 Classification of food flavourings 1035.3 Chemistry of food flavourings 1055.4 Quality control of flavour compounds 1195.5 Analytical methods for the analysis of food flavourings 120


6 Food Acids and Acidity Regulators 1256.1 What are food acids and acid regulators? 1256.2 Types of food acids 1266.3 Uses of food acids 128


7 Food Colour and Colour Retention Agents 1317.1 Why add colourants to foods? 1317.2 Classification of food colourants 1317.3 Overview of colourants 1337.4 Chemistry of food colourants 1437.5 Extraction from natural sources 1437.6 Quality assurance of food colourants 1447.7 Analytical methods 145

References 145

8 Flour Treatment/Improving Agents 1488.1 What are flour treatment/improving agents? 1488.2 Flour maturing agents 1488.3 Flour bleaching agents 1518.4 Flour processing agents 154

References 154

9 Anticaking Agents 1559.1 The caking phenomena 1559.2 Mechanisms of caking 1569.3 Classification of anticaking agents 1599.4 Anticaking agents in use 159


Contents vii

10 Humectants 16210.1 Humectants and moisture control 16210.2 Classification of humectants 162

References 166

11 Antifoaming Agents 16711.1 Sources of foam in food processing 16711.2 Properties of antifoaming agents 16811.3 Mechanisms of antifoaming and foam destabilisation 16811.4 Synthetic defoamers 16811.5 Natural defoamers 170

References 171

12 Minerals and Mineral Salts 17212.1 The importance of minerals and mineral salts 17212.2 Inorganic mineral salts 17312.3 Organic mineral salts 175

References 176

13 Dietary Supplements 17713.1 Introduction to dietary supplements 17713.2 Classification of vitamins 17813.3 Vitamin A (retinols) 17913.4 Vitamin D (calciferol) 18913.5 Vitamin E 19413.6 Vitamin K 19613.7 Vitamin B 19913.8 Vitamin C (L-ascorbic acid) 21013.9 Conclusions 212

References 213

14 Glazing Agents 21814.1 Introduction to glazing agents 21814.2 Mineral hydrocarbon glazes 21814.3 Chemistry of MHCs 22014.4 Conclusion 222

References 223

15 Preservatives 22415.1 Preservatives: Past, present and future 22415.2 Natural food preservatives 22615.3 Traditional food preservation methods 23115.4 Artificial preservative agents 23215.5 Modern food preservation techniques 23515.6 Safety concerns of food preservatives 23715.7 Analytical methods for the determination of preservative residues 23815.8 Conclusions 238


viii Contents

16 Nutraceuticals and Functional Foods 24416.1 What are nutraceuticals? 24416.2 Classification of nutraceuticals 24516.3 Mechanisms of action 24616.4 Conclusion 253


17 Nutritional Genomics: Nutrigenetics and Nutrigenomics 25817.1 Nutrition and gene expression 25817.2 Nutrigenetic areas of application 26017.3 Analytical methods for nutrigenetical food functions 26817.4 Conclusion 270

References 270

18 Probiotic Foods and Dietary Supplements 27418.1 Microbial gut flora activity 27418.2 Probiotics and nutrition 27518.3 Probiotics and health 27518.4 Safety and stability of probiotics 27718.5 Suitable dietary carriers for probiotics 27818.6 Assessment of probiotics in foodstuffs and supplements 27918.7 Conclusions 280

References 281

19 Prebiotics 28519.1 Prebiotics and health 28519.2 Factors that influence the activity and effectiveness of prebiotics 28619.3 Types of oligosaccharides 28619.4 Quality assessment of prebiotics 28919.5 Conclusions 290

References 290

20 Synbiotics 29120.1 Synbiotic foods and health 29120.2 Health benefits of synbiotics 29220.3 Mechanism of action of synbiotics 29320.4 The future of synbotic foods 294

References 294

21 Microencapsulation and Bioencapsulation 29521.1 Introduction to microencapsulation and bioencapsulation 29521.2 Commonly used food-grade microcapsules 29721.3 Methods of food microencapsulation 30321.4 Microencapsulation for food colourants 30721.5 Bioencapsulation for probiotics 30921.6 Conclusions 310

References 310

General Conclusions 314Index 315

Preface

The incorporation of additives in food preparations has been in practice since time immemo-rial. Additives are used to perform various functions, for example, to impart or enhanceflavour (taste) where it is not sharp enough to meet consumer’s demand, to give foodstuffs adesired colour (look/appearance) or to increase the shelf life of the food (preservative role).Some additives perform as essential elements or nutritious supplements to cater for the dietdeficiencies of specific groups of people; without such additives these individuals wouldsuffer from some specific nutrient deficiency syndrome or malnutrition.

The tendency to incorporate additives in food products has increased lately, with theadvent of many new types of additives on the market. Knowledge regarding food additives,how they are prepared, their compositions and how they work has become very importantto those in the food industry and research and academic institutions. This book is thereforeintended to address all these aspects of food additives, and is expected to be of interest to allstakeholders in academia and research.

The book covers the chemistry of selected food additives such as their chemical nature, theway in which they are incorporated in foods and the technology involved in their preparationsand processing steps. The book also covers the mechanisms or modes of action for the activeingredients in each type and class of food additive and preservative; their physico-chemicalcharacteristics which give them special qualities to be used in food processing; parametersused as indicators for the quality assurance of the products; structure-activity relationships;and their safety to consumers.

There has recently been concern about the possible toxicity of some food additives andfood processes. This has led to either a total ban of some additives or maximum limits havebeen set and strict rules have been enforced to safeguard the health of consumer. This aspecthas also been dealt with in this book, and the reported toxic additives are discussed as well asthe analytical methods to determine the safety of various food additives. Standard methodsfor control, monitoring and quality assurance certification for food additives have been set inplace by various regulatory bodies such as the European Union (EU) and the American Foodand Drug Administration (FDA) to control the legality of use for all the additives. Methodsfor the monitoring of additives and their metabolites are also discussed.

The classes of food additives that are discussed in this book include: antioxidants andradical scavengers; emulsifiers; stabilisers, thickeners and vegetable gums; sweeteners; fra-grances, flavourings and flavour enhancers; food acids and acidity regulators; colouringsand colour retention agents; flour treatment/improving agents; anticaking agents; humec-tants; antifoaming agents; minerals and mineral salts; glazers; preservatives; nutraceuticals,nutrigenomics and nutrigenetics; probiotics; prebiotics; synbiotics and micro (bio) capsules.

This book is expected to be a valuable asset to scholars, especially those enrolled inpostgraduate courses and research programs in the areas of food chemistry, food processingand food technology, and also to industrialists and researchers in related areas.

Introduction

Food is one of the main basic human requirements of life and is sourced mainly from plantsor animals (and other minor sources such as fungi e.g. mushroom and algae e.g. Spirulina).Generally, human foods are never consumed raw; rather, they undergo special processingtreatments with or without heat to make them more palatable. The steps involved in the foodprocessing treatments vary depending on the type of food being prepared. Where necessary,some nutritive additives essential for health are added. The process of adding additives infoods involves mixing together various ingredients before or during a heat-treatment step togive the food the intended flavour, taste, texture or appearance. To attain a balanced diet, ithas been necessary to add to certain foodstuffs some ingredients missing in that particulardiet such as salt, amino acids and vitamins. In cases where food is processed for future use orwhere there is a necessity to avoid spoilage by the action of microbes, special treatments suchas smoking or salting are used to keep the food safe for long periods of time. The tendencyto make foodstuffs more appealing and palatable has paved way for the incorporation of avariety of ingredients or some special treatments to impart a desired quality to foodstuffs.This tendency echoes the saying: ‘people first eat with their nose, then with their eyes andfinally with their mouths’. Aroma, flavour, taste and appearance are all equally important inthe appeal of foods.

Food additives are substances incorporated in edible products in order to perform specificroles and functions, such as preservation of foodstuffs by either increasing shelf life orinhibiting the growth of harmful microbes. Other roles include imparting desired colour,odour or a specific flavour to food. Food additives may have a natural origin in the sensethat they may be found existing naturally forming part of the indigenous components of thefood, or they may be synthetic but replicas of substances found naturally in foodstuffs. Theymay also be entirely artificial, which implies that they are synthetically produced and are notcopies of any compounds found in nature.

There are a number of additives and preservatives commonly used in foods includingantioxidants, acids, acid regulators and salts, emulsifiers, colouring agents, minerals andvitamins, stabilisers, thickeners, gelling agents, sweeteners and preservatives. These differentfood additives have different roles to play in foods depending on their intended purpose. Forinstance, emulsifiers tend to give food a good texture as well as good homogeneity suchthat they make it possible for immiscible items such as water and oils to mix well withoutany separation, as is the case in ice-creams or mayonnaise (Suman et al. 2009). Stabilisers,thickeners and gelling agents provide strong texture and smoothness as well as an increasein viscosity (Quemener et al. 2000).

Sweeteners are important as flavours, although there are other types of sweetener flavourswhich perform an important function in the diets of consumers with health problems such asdiabetes (Hutteau et al. 1998).

Nutritive additives such as minerals, vitamins, essential amino acids, etc., are added toparticular food products where they are missing (Nayak and Nair 2003) or in foodstuffs

Introduction xi

specifically intended for people with deficiency of such additives, for example milk forbabies (Ikem et al. 2002). Other additives such as antioxidants are needed for the preventionof fat and oil rancidity in baked foods by inhibiting the effects of oxygen on foods and alsopreventing the loss of flavour, thereby maintaining food palatability and wholesomeness.

Acids and acidic regulators such as citric acid, vinegar and lactic acid are food additivesto control food pH (levels of acidity or alkalinity) and they play an important role in thesharpening of flavours (Populin et al. 2007), as preservative (Brul and Coote 1999) and as an-tioxidants. Some acids and acid regulators tend to release acids only when they are subjectedto a heat treatment such as with some bakery products (e.g. acids produced by the leaveningagents react with baking soda to make the bakery products rise during the baking process).

Colouring and colour retention agents are added to foods to appease the eye of theconsumer or beholder; they are also intended to maintain the colour of food in cases whereit may fade (MacDougall 1999).

Generally speaking, the desire for a particular quality of food has resulted in the intro-duction of numerous additives with wide applications in different cultures and civilisations.Currently, many different types of food additives have been commercialised and are findingtheir way onto the markets worldwide (Baker 2010). This trend in business has contributed tothe speedy growth in food processing and other related industries, where food additives areused en masse. The economic success of food additives has further encouraged the adventof new technologies in the processing of foods.

However, these new technologies and additives have brought other unwanted outcomesand are an issue of concern. Despite all the benefits and advantages of food additives andpreservatives, there is still a potential danger of chemical adulteration of foods. Additives orpreservatives in foods may themselves trigger other hormonal or chemical processes in thebody that can generate negative physiological responses. The metabolites produced by addi-tives may also cause side effects, because not all food additives enter the markets after beingthoroughly studied to prove their safety (Skovgaard 2004). Although most food additivesare considered safe, some are known to be carcinogenic or toxic. For these reasons, manyfood additives and preservatives are controlled and regulated by national and internationalhealth authorities. All food manufacturers must comply with the standards set by the relevantauthorities without violating the maximum thresholds stated to ensure the safety of the finalproduct to the consumers. In most cases, food processing industries must seek standard certi-fication before using any new additive or preservative or before using any originally certifiedadditive or preservative in a different way (Pinho et al. 2004; Skovgaard 2004).

REFERENCES

Baker, S. R. (2010) Maximizing the use of food emulsifiers. MSc thesis, Kansas State University, Manhattan,Kansas, USA.

Brul, S. & Coote, P. (1999) Preservative agents in foods: Mode of action and microbial resistance mechanisms.International Journal of Food Microbiology 50, 1–17.

Hutteau, F., Mathlouthi, M., Portmad, M. O. & Kilcast, D. (1998) Physicochemical and psychophysicalcharacteristics of binary mixtures of bulk and intense sweeteners. Food Chemistry 63 (1), 9–16.

Ikem, A. Nwankwoala, A., Odueyungbo, S., Nyavor, K. & Egiebor, N. (2002) Levels of 26 elements ininfant formula from USA, UK, and Nigeria by microwave digestion and ICP–OES. Food Chemistry 77,439–447.

MacDougall, D. B. (1999) Coloring of Food, Drugs, and Cosmetics. Marcel Dekker, Inc., New York, Basel,USA.

xii Introduction

Nayak, B., & Nair, K. M. (2003) In vitro bioavailability of iron from wheat flour fortified with ascorbic acid,EDTA and sodium hexametaphosphate, with or without iron. Food Chemistry 80, 545–550.

Pinho, O., Ferreira, I. M. P. L. V. O., Oliveira, M. B. P. P. & Ferreira, M. A. (2000) Quantitation of syntheticphenolic antioxidants in liver pates. Food Chemistry 68, 353–357.

Populin, T., Moret, S., Truant, S. & Conte, L. S. (2007) A survey on the presence of free glutamic acid infoodstuffs, with and without added monosodium glutamate. Food Chemistry 104, 1712–1717.

Quemener, B., Marot, C., Mouillet, L., Da Riz, V. & Diris, J. (2000) Quantitative analysis of hydrocolloids infood systems by methanolysis coupled to reverse HPLC. Part 1. Gelling carrageenans. Food Hydrocolloids14, 9–17.

Skovgaard, N. (2004) Safety evaluation of certain food additives and contaminants. International Journal ofFood Microbiology 90, 115–118.

Suman, M., Silva, G., Catellani, D., Bersellini, U., Caffarra, V. & Careri, M. (2009) Determination of foodemulsifiers in commercial additives and food products by liquid chromatography/atmospheric-pressurechemical ionization mass spectrometry. Journal of Chromatography A, 1216, 3758–3766.

List of Abbreviations

AAPH 2, 2′-azobis (2-amidino-propane) dihydrochlorideABTS 2, 2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)ACI amylose complexing indexAEDA aroma extract dilution analysisAMG acetylated monoglycerideAPCI-MS atmospheric pressure chemical ionisation mass spectrometryAV acid valueBDMS butyldimethylsilylBHA butylated hydroxyanisoleBMI body mass indexBR Brigg–RauscherCDG calcium diglutamateCE capillary electrophoresisCMG citrate monoglyceridesCSL calcium stearoyl 2 lactateCTAB cetyltrimethylammonium bromideCTAC cetyltrimethylammonium chlorideCZE capillary zone electrophoresisDAD diode array detectorDHC dihydrochalconeDMPD N,N-dimethyl-p-phenylenediamineDPPH 1, 1-Diphenyl-2-picrylhydrazylEDTA ethylenediaminetetraacetic acidELISA enzyme-linked immunosorbent assayEU European UnionFACE fluorophore-assisted carbohydrate electrophoresisFAO Food and Agriculture OrganizationFDA Food and Drug AdministrationFRAP ferric-reducing ability of plasmaFT-IR Fourier transform infrared spectrometryGC gas chromatographyGDL glucano-delta-lactoneGI glycemic indexGL glycemic loadGLC gas liquid chromatographyGPC gel permeation chromatographyGRAS generally recognised as safeHDB hexadimetrine bromide

xiv List of Abbreviations

HFCS high-fructose cone syrupHHP high hydrostatic pressureHIL high-intensity laserHLB hydrophilic–lipophilic balanceHORAC hydroxyl radical antioxidant capacityHPAEC high-performance anion exchange chromatographyHPH high-pressure homogenisationHPLC high-performance liquid chromatographyHPU high-power ultrasoundHVAD high-voltage arc dischargeLDL low-density lipoproteinLOD limit of detectionLOQ limit of quantificationMALDI-MS matrix-assisted laser desorption-ionisation mass spectrometryMAP modified atmosphere packagingMEKC micellar electrokinetic chromatographyMSG monosodium glutamateNNS non-nutritive sweetenersOAV odour activity valuesOMF oscillating magnetic fieldsORAC oxygen radical absorbance capacityPCL photochemiluminescencePEF pulsed electrical fieldsPG propylene glycolPGA propylene glycol alginatePGPR polyglycerol polyricinoleatePHIL pulsed high-intensity lightPKU phenylketonuriaPPO polyphenol oxidasePWL pulsed white lightRMCD random methylated b-cyclodextrinRNS reactive nitrogen speciesROS reactive oxygen species-SH sulphhydrylSMG succinylated monoglycerideSOD superoxide dismutaseSP streamer plasmaSWV square-wave voltammetryTBARS thiobarbituric acid reactive substancesTEAC trolox equivalent antioxidant equivalentTMS trimethylsilylTRAP total radical trapping antioxidant parameterTSS total soluble solidsUV-Vis ultraviolet-visibleWHO World Health Organisation

1 Antioxidants and Radical Scavengers

Abstract: Food antioxidants play an important role in the food industry due to their abilityto neutralise free radicals that might be generated in the body. They do that by donatingtheir own electrons to free radicals without becoming free radicals in the process themselves,hence terminating the radical chain reaction. The converted free radical products will thenbe eliminated from the body before causing any harm; in this regard, antioxidants play therole of scavengers protecting body cells and tissues. In this chapter, the processes which leadto the formation of these reactive species (free radicals) and the different additives used asantioxidants or radical scavengers to counter the effects of free radicals will be discussed.Sources of different types of antioxidants, the various mechanisms by which they work andanalytical methods for determination and quality control are also examined.

Keywords: antioxidants; free radical species; ORAC assay; HORAC assay; DPPH assay;FRAP assay; Trolox; TEAC assay; ABTS assay; PCL assay; DMPD assay; DL assay;TBARS assay; Brigg-Rauscher assay

1.1 CHEMISTRY OF FREE RADICALS AND ANTIOXIDANTS

1.1.1 Introduction

From the viewpoint of chemistry, free radicals refer to any molecule with an odd unpairedelectron in its outer electronic shell, a configuration responsible for the highly reactive natureof such species. The presence of such highly reactive free radicals in biological systems isdirectly linked to the oxidative damage that results in severe physiological problems. Thefree radical species that are of concern in living systems include the reactive oxygen species(ROS), superoxide radicals (SOR), hydroxyl radicals and the reactive nitrogen species (RNS).The oxygen-containing reactive species are the most commonly occurring free radicals inliving medium and are therefore of greatest concern. The oxidative damage caused by thesefree radicals can be prevented by using antioxidants which include enzymatic antioxidantsystems such as catalase, glutathione peroxidase and superoxide dismutase (SOD) as wellas non-enzymatic antioxidants (Figure 1.1). It should be noted that, in nature, the generationof free radicals which cause oxidative stress and that of antioxidants or radical scavengers iscarefully controlled such that there is always a balance between the two (Vouldoukis et al.2004). Examples of non-enzymatic antioxidants include vitamin C (ascorbic acid) whichis a sugar acid, vitamin E (�-tocopherol) and �-carotene, bilirubin, propyl gallate (PG, a

Chemistry of Food Additives and Preservatives, First Edition. Titus A. M. Msagati.C© 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

2 Chemistry of Food Additives and Preservatives

OH

CC

CH3

CH3

H3C

H3C

H3C

CH3

CH3

OH

C

O

H3CCH3

CH3

CH3

(a) (b)

OH

C

OH

CH3

H3C CH3

OH

OH

HO

C

OO

C3H7

(c)

(d)

HC

HO

OHCH

CH

CH

OH

OH

HO

HO CH3

H3CH3C

CH3

O

O

H3C CH3

(e)

OCH3

(CH2)3

CH3

H3C

HO

CH3

CH

CH3

(CH2)3 CH

CH3

(CH2) 3CH

CH3

CH3

(f)

Fig. 1.1 Examples of synthetic antioxidants used in food industries: (a) BHT; (b) BHA; (c) t-BHQ; (d) PG;(e) gossypol; and (f) tocopherol.

condensation ester product of gallic acid and propanol), uric acid, tertiary butylhydroquinone(t-BHQ), butylated hydroxyanisole (BHA), ubiquinone and macromolecules which includeceruloplasmin, albumin and ferritin. Generally, mixtures of different antioxidants providebetter protection against attack by free radicals rather than individual antioxidants.

Due to the importance of antioxidant systems, there are a number of quality assess-ment criteria for the antioxidant performance of these systems. Various assays have been

Antioxidants and Radical Scavengers 3

developed to assess the antioxidant capacities, including the oxygen radical absorbancecapacity (ORAC) assay, ferric reducing ability of plasma (FRAP), Trolox equivalent antiox-idant capacity (TEAC) assay, etc. Antioxidant foods which are dietary nutrients containingantioxidant compounds and non-nutrient antioxidants which are normally added to foods toplay the role of antioxidants will be discussed simultaneously in this chapter, unless indicatedotherwise.

Further Thinking

Free radicals are undesirable due to their instability caused by the electron deficienciesin their structures. They have a high electronic affinity which makes them attack anymolecule in their vicinity, generating a chain of reactions which are detrimental to thebody and which instigate disorders, diseases, aging and even death.

1.1.2 The formation of ROS in living systems

Under normal conditions, oxygen is vital in metabolic reactions which are necessary for life.Due to its high reactive nature however, oxygen also causes severe damage to living systemsdue to the generation of reactive oxygen species (ROS; Davies 1995).

The reactive free radicals are generated as part of the energy generation metabolic pro-cesses (Raha and Robinson 2000), and are released as a result of a number of reactionprocedures in the electron transport chain as well as in the form of intermediate reductionproducts (Lenaz 2001). Due to the highly reactive nature of free radicals that are formed asintermediates, they prompt electrons to proceed in a concerted fashion to molecular oxygenand thus generate superoxide anion (Finkel and Holbrook 2000). A similar scenario occursin plants for example, whereby reactive oxygen species are produced during the process ofphotosynthesis (Krieger-Liszkay 2005).

Examples of reactive species produced as a result of these metabolic reactions include:superoxide anion (O2

−), hydrogen peroxide (H2O2), hypochlorous acid and hydroxyl radical(·OH) (Valko et al. 2007). The hydroxyl radicals are known to be unstable; they react spon-taneously with other biological molecules in a living medium, causing destructive reactionsin foodstuffs and serious physiological damage to consumers (Stohs and Bagchi 1995).

1.1.3 Negative effects of oxidants in food processesand to food consumers

The oxidation process brings about destructive reactions in food items that lead to off-flavourand loss of colour and texture due to the degradation of carbohydrate, protein, vitamins,sterols and lipid peroxidation (Hwang 1991; Pinho et al. 2000; Kranl 2004). The conse-quences to consumers include damage to nucleic acids, cellular membrane lipids and othercellular organelles, carcinogenesis, mental illnesses and disorders, lung diseases, diabetes,atherosclerosis, autoimmune diseases, aging and heart diseases (Finkel and Holbrook 2000;Lachance et al. 2001; Ou et al. 2002; Yu et al. 2005; Nakabeppu et al. 2006).


1.1.4 Reactive oxygen/nitrogen species and aging

There is strong scientific evidence which relates the reactive oxygen/nitrogen species(ROS/RNS) to aging and pathogenesis (Lachance et al. 2001; Yu et al. 2005; Nakabeppuet al. 2006). In addition, facts have also been presented in many scientific reports that ROSsuch as peroxyl radicals (ROO·), superoxide ion (O2·+), hydroxyl radicals (HO), etc. playan active role in promoting or inducing numerous diseases such as different types of cancers(Finkel and Holbrook 2000; Ou et al. 2002). Unless these adverse reactions are retardedor prohibited, they will result in food deterioration and health problems to consumers. Tocounter such harmful effects, antioxidants have been incorporated in many foodstuffs tominimise or solve the problem altogether.

Further Thinking

The incorporation of antioxidants in foodstuffs serves a number of purposes, includingthe prevention of rancidity phenomena as a result of oxidation (which results in badodour and off-flavour) of food items containing fats and oils. Antioxidants are alsoessential in the retention of the integrity of food items (mainly fruits, fruit juices andvegetables) because of their particular properties in preventing browning reactions,extending the shelf life of these food items.

1.2 TYPES OF ANTIOXIDANTS

Antioxidants as food additives are used to delay the onset of or slow the pace at which lipidoxidation reactions in food processing proceed. Most of the synthetic antioxidants contain aphenolic functionality with various ring substitutions (monohydroxy or polyhydroxy phenoliccompounds) such as butylated hydroxytoluene (BHT), BHA, t-BHQ, PG, gossypol andtocopherol (Figure 1.1). These compounds make powerful antioxidants to protect foodstuffsagainst oxidative deterioration of the food ingredients. The main chemical attribute thatmakes them suitable as antioxidants is their low activation energy property, which enablesthem to donate hydrogen easily and thus put on hold or lower the kinetics of lipid oxidationmechanisms in food systems. The delay to the onset or slowing of the kinetics of lipidoxidation is possible due to the ability of these compounds to either block the generationof free alkyl radicals in the initiation step or temper the propagation of the free radicalchain. Due to their positive effects in food processes antioxidants are also known as potentialtherapeutic agents, thus playing a medicinal role as well. For safety purposes and adherence toquality control standards, the use of any synthetic antioxidant preparation in food processesis expected to meet the following criteria: effective at low concentrations; without anyunpleasant odour, flavour or colour; heat stable; non-volatile; and must have excellent carry-through characteristics (Shahidi and Ho 2007).

1.2.1 Natural antioxidants of plant origin

In addition to chemical or synthetic antioxidants, there are also a number of antioxidants thatexist naturally in plants and many other herbal materials (Shahidi and Naczk 1995).


Plants that contain natural antioxidants include: carrots, which contain �-carotene andxanthophyll (Chu et al. 2002); ginger roots (Halvorsen et al. 2002); and citrus fruits withtheir abundance of flavonoid compounds and ascorbic acid (vitamin C) (King and Cousins2006). Tomatoes and pink grapefruit contain ascorbic acid and other carotenoid compoundsknown as lycopenes which are antioxidants (King and Cousins 2006). Grape seeds well astheir skin extracts also contain a number of antioxidant substances, mainly proanthocyani-din bioflavonoids and tannins (DerMarderosian 2001). Saccharomyces cerevisiae, whichis also known as nutritional yeast, has antioxidants superoxide dismutase (SOD) and glu-tathione (King and Cousins 2006). Green tea is also known to be rich in catechins andother polyphenol antioxidants (Cai et al. 2002; Thielecke and Boschmann 2009); vegetableoils such as soybean oil contains radical scavengers such as vitamin E (tocopherols andtocotrienols) (Nesaretnam et al. 1992; Beltran et al. 2010); legumes such as soybean areknown to be rich in isoflavones (Luthria et al. 2007); oil seeds such as canola and mustardcontain phenolic acids and phenylpropanoid antioxidants (Shahidi and Wanasundara 1995);and cereals such as wheat contains phenolic and other flavonoid radical scavengers (Shenet al. 2009).

Further Thinking

In nature there are many different types of foodstuffs which are known to be rich inantioxidants. Examples include fruits (grape, orange, pineapple, kiwi fruit, grape-fruit, etc.), vegetables (cabbage, spinach, etc.), cereals (barley, millet, oats, corn,etc.), legumes (beans, soybeans, etc.) and nuts (groundnuts, peanuts, etc.). Daily in-take of a variety of these antioxidant foods may bring significant health benefits toconsumers.

1.2.2 Phenolic non-flavonoid antioxidant compoundsfrom natural sources

Polyphenolic non-flavonoid antioxidant compounds include resveratrol and gallic acid whichare abundant in plants such as tea, grapes (red wine) and a variety of other fruits (Amakuraet al. 2000; Rechner et al. 2001). Resveratrol, a phenolic non-flavonoid compound ex-tract from wine, has been reported to inhibit low-density lipoprotein oxidation and reduceplatelet aggregation, hence playing a direct role in combating atherothrombogenesis (Frankelet al. 1995; Pace-Asciak et al. 1995; Belguendouz et al. 1997). Resveratrol is consideredan important agent for the cardio-protective action of wine and also plays an importantrole in reducing hepatic synthesis of cholesterol and triglyceride, as observed in experi-ments performed in rats (Arichi et al. 1982; Hung et al. 2000). It also inhibit the synthe-sis of eicosanoids and rat leukocytes, interfering arachidonate metabolism (Kimura et al.1985a, b), and inhibits the activity of some protein kinases (Jayatilake et al. 1993). All thesebiological and pharmacological activities of resveratrol are due to its antioxidant property(Rimando et al. 2002). The polyphenolic compound gallic acid (3,4,5-trihydroxybenzoicacid) (Figure 1.2), obtained naturally as a product of either alkaline or acid hydrolysis oftannins, and its derivatives is also found abundantly in wine (Aruoma et al. 1993).


OHO

OH

OH

HO

HO

HO

OH

O

OH

HO

OH

OH

O

OH

O

OH

OH

OH

H

HO OH

H H

O OCH2

HO

H

OHH

CH3

O

HO

O

Quercetin

Rutin

Gallic acid

Trans-resveratrol

Fig. 1.2 Chemical structures of phenolic non-flavonoid antioxidants.

1.2.3 Phenolic flavonoid antioxidant compoundsfrom natural sources

Antioxidants with flavonoid functionality are low-molecular weight polyphenolics whichoccur in a variety of vegetables and fruits (Hertog et al. 1992). An example of these flavonoidpolyphenolic compounds is quercetin, which forms the main aglycone found in many foods(Robards et al. 1999). Apart from functioning as antioxidants, various flavonoids also haveanti-inflammatory, anti-allergic, anticancer and anti-hemorrhagic properties (Das 1994). Theantioxidant properties of flavonoids are responsible for the protective effect of wine andvegetable-rich diets against coronary heart disease (Pearson et al. 2001). The majority of


phenolic flavonoids extracted from natural sources (for example, gallic acid, trans-resveratrol,quercetin and rutin; Figure 1.2) have demonstrated potential beneficial effects on humanhealth in many ways.

1.2.4 Acidic functional groups responsiblefor antioxidant activity

The antioxidant activity of certain food plants are due to various functional groups associ-ated with some organic acids such as vanillic, ferulic and p-coumaric acids, found mainlyin whole grains. Other acids found in barley grains such as salicylic, p-hydroxybenzoic,protocatechuic, syringic and sinapic acids have functional groups that confer antioxidantactivity (Shahidi and Naczk 1995). Generally, corn wheat and barley contain syringic acid,sinapic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, ferulic acid, salicylicacid and p-coumaric acid as molecules containing antioxidant functional groups (Figure 1.3;Hernandez-Borges et al. 2005).

Further Thinking

Who needs antioxidants and why?� Children need lots of antioxidants (�-carotene, flavonoids, vitamins C and E) as

damage caused by free radicals has a much greater effect on their young and tenderbodies than compared to adults. Some antioxidants are added to infant formulas(e.g. ascorbyl palmitate, tocopherols and lecithin).

� The elderly need antioxidants since the oxidative damage due to free radicals affectsthe performance of muscles to a greater degree with age, affecting the physicalperformance and reducing fitness in many areas.

� Active sportsmen and those who take part in strenuous exercise or heavy workinvolving massive physical muscle energy need more antioxidants to protect againstthe by-products of exercise. This group need extra fatty esters and antioxidants fromdiets including spices such as from plants of Curcuma longa L. and Zingiberaceae,or collastin supplements which contain natural cyclooxygenase-2 inhibitors that arecapable of protecting against cell damage as well as inflammation. Diets with theseingredients as well as some specific antioxidants are essential in maintaining bodyjoints, thus keeping sportsmen fit.

� Healthy people need antioxidants as protection from various diseases, illnesses andsicknesses such as cancer, diabetes, etc.

1.3 EFFICACY OF DIFFERENT ANTIOXIDANTS

The compositions, structural features and chemical structures of antioxidants are importantparameters that control their efficacy and also the antioxidant activity (Bors et al. 1990a,b). For example, the presence of ortho-dihydroxy functionality in the catechol structure offlavonoid antioxidants has been associated with the increased stability of radicals generateddue to the possible formation of hydrogen bonding or the delocalisation of electrons around


OH

OH

HO

O OH

OOH

OOH

OHO

OH

HOO

OOH

O

OH

HO

OH3C

H3C

Vanillic acid syringic acid

OH

O

HO

p-coumaric acid

OH

O

HO

OH3C

OH

O

HO

OH3C

OH3C

Ferulic acid

Sinapinic acid

Fig. 1.3 Chemical structures of some antioxidants with acidic functional groups.


the aromatic ring (Apak et al. 2007). The presence of hydroxyl groups at positions 3 and 5 ofphenolic antioxidants is said to contribute to the stability of antioxidants (Firuzi et al. 2005).Phenolic compounds which are dihydroxylated or hydroxylated at position 2 or 4 (orthoor para) or contain a methoxy group are generally more effective than simple phenolics(Van Acker et al. 1996; Apak et al. 2007; Bracegirdle and Anderson 2010). This is due tothe presence of methoxy groups in ortho and para positions of the ring serving as electron-donating groups, thus adding to stability and hence promoting the antioxidant activity (Firuziet al. 2005).

Moreover, phenylpropanoid antioxidants with extended conjugation are known to haveenhanced antioxidant activity compared to benzoic acid derivatives because of the resonancestabilisation. The hydrophilicity as well as lipophilicity of the antioxidants is dependent onthe correct matching in terms of application of antioxidants; more hydrophilic antioxidantsmatches is best for use in stabilising bulk oil systems as opposed to oil-in-water emulsions,while the converse is true for the activity of lipophilic antioxidants (Shahidi and Ho 2000).

Further Thinking

Unsaturated and polyunsaturated fats may be preferred over saturated animal fats bymany. However, polyunsaturated and saturated fats undergo oxidation easily, hencethe problem of rancidity due to the decomposition of fat when they react with oxygen.Peroxides are produced, which result in a bad smell, off-flavour (rancidity) and thesoapy texture of food. If oxidation reactions occur in the body system they cause fatdeposits to be built up, which may block blood vessels. This necessitates the incor-poration of antioxidants in foods which may react with oxygen, hence preventing theformation of peroxides as well as heart problems, cancer diseases, arthritis, tumoursetc. Antioxidants also help to preserve the integrity of food items so that they remainfit for human consumption for a long time.

1.4 ACTION MECHANISMS OF ANTIOXIDANTS

From the definition of an antioxidant compound – which refers to a chemicals speciescapable of suppressing the harmful effects of reactive radicals present in biological systemsat low concentration (Gutteridge 1994) – it follows that the mechanisms should involve theprotonation by the donor species to the reactive radicals. There are a number of possiblemechanisms for antioxidant action and these include: (1) quenching mechanism, whichoccurs when the radical is in an excited triplet state which makes the antioxidant behave as aquenching agent (Tournaire et al. 1993; Anbazhagan et al. 2008; Ji and Shen 2008); (2) directhydrogen transfer mechanism which takes place if the radical is in a doublet state, enablingthe direct transfer of the hydrogen atom to the radical (Priyadarsini et al. 2003; Luzhkov2005); (3) charge transfer for doublet radical which yields a closed-shell anion and a radicalantioxidant cation (Kovacic and Somanathan 2008; Oschman 2009); and (4) bond-breakingmechanisms, as in the case for vitamin E (Graham et al. 1983; Roginsky and Lissi 2005).


1.4.1 Quenching

In this mechanism, which is also known as singlet oxygen scavenging, antioxidants reactswith singlet oxygen (1O2) to form intermediate compounds such as endoperoxides andfinal products which are mainly hydroperoxydienones. The final products are responsiblefor quenching, that is, termination of the propagation process that generates free radicals.Examples of antioxidants which exhibit this phenomenon include vitamin E and carotene.

1.4.2 Hydrogen transfer

A complex is formed between a lipid radical and the antioxidant radical which, in this case,is the free radical acceptor. The processes involve several reactions as depicted in Figure 1.4.

1.4.3 Charge transfer

There are two ways in which the charge transfer antioxidation mechanism takes place,both involving the formation of stable radicals which stops the propagation of reactivespecies in the biological systems. Firstly, the antioxidation mechanism may occur through

+ Lipid radicals

OH

C(CH3)3

OCH3

C.C(CH3)3

OCH3

+ Hydrogenatedradicals

O

C(CH3)3

OCH3

.

.OCH3

C(CH3)3

O

O

C(CH3)3

OCH3

.

Butylated hydroxyanisole

Stable radicals ofbutylated hydroxyanisole

Fig. 1.4 Possible mechanism of butylated hydroxyanisole antioxidants (Lambert et al. 1996; Goodmanet al. 1990)


O

HO

H3C

CH3

CH3

CH3 CH3

CH3

CH3

O

CH3

H3C

O

[O2]

CH3

H3C

HO

CH3 CH3

CH3

CH3

α-tocopherol

α-tocoquinone

Fig. 1.5 Possible mechanistic reaction of �-tocopherol antioxidant (Herrera and Barbas 2001).

hydrogen transfer processes in which the reactive species themselves abstract a proton fromthe antioxidant, such that the antioxidant will become a highly stable radical which cannotreact with any substrate. The stability of this stable radical is enhanced by resonance effectsand hydrogen bonding. The second mechanism is by a one electron transfer process wherethe antioxidant can donate an electron to the reactive species, making itself a highly stablepositively charged radical which cannot undergo any reaction with substrates. Examplesof antioxidants which undergo charge transfer mechanisms include flavonoids and otherphenolic antioxidants.

1.4.4 Bond-breaking

The �-tocopherol (Figure 1.5) is a hydrophobic antioxidant which plays an important role inprotecting the cytoplasmic membranes against oxidation reactions caused by lipid radicals. Itprotects cell membranes by reacting with the lipid radicals, thus terminating the chain prop-agation reactions due to the reactive species that would otherwise have continued oxidationreactions with the cell membrane (Herrera and Barbas 2001).

1.5 STRUCTURE–ACTIVITY RELATIONSHIPOF ANTIOXIDANTS

1.5.1 Polyphenol antioxidants

With the phenolic antioxidants it has been established that the presence of o-dihydroxystructure in the B ring (Figure 1.6) contributes significantly to the higher stability of theradical; it also plays a significant role in electron delocalisation, necessary for the antioxidantactivity. Moreover, the 3- and 5-OH groups with 4-oxo function in the A and C rings have


OHO

OH O

OH

OHO

OH

OH

OHO

OH

OH

OH

OH

OH

OH

OH

OH

B

CA

QuercetinCatechin

Cyanidin

A C

B

A C

B

2

35

6

7

8

3'2'

4'

5'

6'

Fig. 1.6 Structure of polyphenol antioxidants.

been reported as necessary for efficient antioxidant activity (Rice-Evans et al. 1996). Theposition and degree of hydroxylation is another aspect that has been reported as essential forthe antioxidant activity of phenols and particularly the o-dihydroxylation of the B ring, thecarbonyl at position 4, and a free hydroxyl group at positions 3 and/or 5 in the C and A rings,respectively.

1.5.2 Flavonoid antioxidants

The activity of flavonoid antioxidants (for example flavones, isoflavones and flavanones)against peroxyl and hydroxyl radicals (pro-oxidants) was studied by Cao et al. (1997). Theyfound that the pro-oxidant activities of these flavonoid antioxidants were strongly influencedby the number of hydroxyl substitutions in their backbone structure, which lacked both theantioxidant as well as the pro-oxidant property. It was evident that the greater the numberof hydroxyl substitutions, the stronger the antioxidant and pro-oxidant activities. It was alsoconcluded that those flavonoids with multiple hydroxyl substitutions had higher antiperoxylradical activities compared to others such as �-tocopherol. Another important observationwas that the presence of a single hydroxyl substitution at position 5 as well as the conjugationbetween rings A and B (Figures 1.7a–c ) provided no activity at all, but the di-OH substitutionat 3′ and 4′ (Figure 1.7b) proved to be essential for the peroxyl radical absorbing activity ofa flavonoid. Cao et al. also studied the effect of O-methylation of the hydroxyl substitutionsand found that it resulted in the inactivation of both the antioxidant and the pro-oxidantactivities of the flavonoids (Cao et al. 1997).


O

O

OH

HO

OH

O OHHO

OH

Flavonol

(a)

Iso-flavonol

2

345

6

7

8

2'

4'

3'

5'

6'

O

A

B

2

3

4

5

6

2'

3'

4'

5'

6'O

O

O

O

B

CA

A C

B

A C

O

O

Flavone

Flavanone Isoflavone

O

O

OOH

HO

OH OCoumarin

(b)Anthocyanin

Fig. 1.7 (a) General structures of the main classes of flavonoid antioxidants and flavonoid-related com-pounds; (b) basic structure of flavonoids; and (c) possible mechanism of flavonoid antioxidants with radicalscavengers (R·) (Pereira and Das 1990).


OH

OH

O

OH

O

O

O

RH

RHR

R

(c)

Fig. 1.7 (Continued)

1.5.3 Mechanism of reactions of flavonoid antioxidantswith radical scavengers

Pereira and Das (1990) have reported that the presence of carbonyl group at C-4 and a doublebond between C-2 and C-3 are important features for high antioxidant activity in flavonoids(see the basic structure of flavonoids, Figures 1.7b and c).

1.6 FACTORS AFFECTING ANTIOXIDANT ACTIVITY

There are a number of physical factors that influence the activity of the antioxidant, discussedin the following sections.

1.6.1 Temperature

Temperature catalyses the acceleration of the initiation reactions, which results in a decreasein the activity of the already-available or introduced antioxidants (Pokorny 1986). Because ofthis, the variations in the temperature normally influence the manner in which some oxidantswork; note that these variations are not the same for all antioxidants (Yanishlieva 2001).For instance, the effect of temperature variations on the activity of different antioxidantsin fats and oils over a large temperature range was that the �-tocopherol activity increasedas the working temperature increased throughout the whole temperature range (20–100◦C)(Marinova and Yanishlieva 1992, 1998; Yanishlieva and Marinova 1996a, b). Another ob-servation on the effect of temperature variation on the antioxidant activity was that some ofthe tested antioxidants were found to be sensitive to either concentration or the stabilisedsubstrate (Marinova and Yanishlieva 1992, 1998).

1.6.2 Activation energy and redox potential

Different antioxidants will have different activation energies as well as oxidation-reductionpotentials. These properties mean that antioxidants have a varying ability to donate anelectron easily.

chemistry of food additives and preservatives chemistry of ...€¦ · 13.5 vitamin e 194 13.6...

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